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Anti-blood coagulation rodenticides, such as warfarin, have been used all over the world. They inhibit vitamin K epoxide reductase (VKOR), which is necessary for producing several blood clotting factors. This inhibition by rodenticides results in lethal hemorrhage in rodents. However, heavy usage of these agents has led to the appearance of rodenticide-resistant rats. There are two major mechanisms underlying this resistance, i.e., mutation of the target enzyme of warfarin, VKOR, and enhanced metabolism of warfarin. However, there have been few studies regarding the hepatic metabolism of warfarin, which should be related to resistance. To investigate warfarin metabolism in resistant rats, in situ liver perfusion of warfarin was performed with resistant black rats (Rattus rattus) from Tokyo, Japan. Liver perfusion is an in situ methodology that can reveal hepatic function specifically with natural composition of the liver. The results indicated enhanced hepatic warfarin hydroxylation activity compared with sensitive black rats. On the other hand, in an in vitro microsomal warfarin metabolism assay to investigate kinetic parameters of cytochrome P450, which plays a major role in warfarin hydroxylation, the V-max of resistant rats was slightly but significantly higher compared to the results obtained in the in situ study. These results indicated that another factor like electron donators may also contribute to the enhanced metabolism in addition to high expression of cytochrome P450.

BACKGROUND Rodenticide resistance to anticoagulants in Rattus norvegicus will lead to increased difficulties in combating these pest animals. Here, the authors present the results of a survey in the Netherlands where tissue samples and droppings were tested using a newly developed TaqMan PCR test for genotypic variation at codon 139 in the Vkorc1 gene associated with anticoagulant rodenticide resistance. Test results are linked to results of a questionnaire that was conducted among pest controllers. RESULTS Genetic mutations at codon 139 of the Vkorc1 gene in R. norvegicus can be encountered in many parts of the Netherlands. In 34/61 rat tails, a genotype was found that is linked to anticoagulant rodenticide resistance (56%). In droppings, 42/169 samples (25%) showed a resistance-mediating genotype. In addition, indications of a clear genetic substructure in the Netherlands were found. In some regions, only resistance-mediating genotypes were found, corroborating results from the questionnaire in which pest controllers indicated they suspected resistance to anticoagulant rodenticides. CONCLUSION This is the first study to demonstrate the presence of multiple genetic mutations at codon 139 of the Vkorc1 gene in R. norvegicus in the Netherlands. As rodenticides should keep their efficacy because they are a last resort in rodent management, more studies are urgently needed that link specific genetic mutations to the efficacy of active substances. © 2014 Society of Chemical Industry

Vitamin K is an essential co-factor in the activation of several coagulation factors and plays an important role in blood coagulation. Resistance against anticoagulant rodenticides is based on a single nucleotide polymorphism in the VKORC1 gene. A pleiotropic effect of rodenticide resistance is an increased need for vitamin K. Therefore we tested homozygous resistant rats (Y139F, L120Q) for their vitamin K requirement by feeding six males and six females of each strain with a vitamin K deficient diet (Altromin) for two weeks, yet coprophagy was not prevented. Then on day 0,3,5,7,10,14 of the experiment prothrombin time (PT) was measured and expressed as international normalized ratio (INR). Rats with an INR>5 were considered as responders. Three L120Q-males showed a slightly increased PT (INR>2) on day 0, while five L120Q-males reached an INR>5 (1/day3, 2/day5, 1/day10 and 1/day14). Within the group of the L120Q-females two rats responded to the treatment respectively on day 10 and 14, and one rat died on day 7 without bleeding signs. One Y139F-male showed an INR>5 on day 7 and two Y139F-males died on day 10 with signs of severe anaemia and so considered as responders. None of the Y139F-females reached an INR>5. According to this preliminary study the L120Q-strain is more sensitive to vitamin K deprivation than the Y139F-strain. Although both strains showed a prolonged clotting time compared to warfarin susceptible strains, it remains unclear in which degree vitamin K deficiency occurs in the field and could affect the relative fitness of resistant rats.

Development and application of a quick test for resistance of brown rats against rodenticides. Resultaten van een onderzoek naar het voorkomen van genetische mutaties bij bruine ratten in Nederland.

Development and application of a quick test for resistance of brown rats against rodenticides.

Resistentie kan een oorzaak zijn voor het falen van een chemische bestrijding van een bruine rattenpopulatie. Men spreekt van resistentie op het moment dat ratten wel rodenticiden opnemen, maar er, door een overerfbaar afgenomen gevoeligheid, niet meer aan komen te overlijden of pas na langere tijdsduur. Maar hoe onstaat resistentie? En wat is er bekend over resistentie voor anticoagulantia bij bruine ratten in Europa?

Resistentie kan een oorzaak zijn voor het falen van een chemische bestrijding van een bruine rattenpopulatie. Men spreekt van resistentie op het moment dat ratten wel rodenticiden opnemen, maar er, door een overerfbaar afgenomen gevoeligheid, niet meer aan komen te overlijden of pas na langere tijdsduur. Maar hoe onstaat resistentie? En wat is er bekend over resistentie voor anticoagulantia bij bruine ratten in Europa?

Resistentie kan een oorzaak zijn voor het falen van een chemische bestrijding van een bruine rattenpopulatie. Men spreekt van resistentie op het moment dat ratten wel rodenticiden opnemen, maar er, door een overerfbaar afgenomen gevoeligheid, niet meer aan komen te overlijden of pas na langere tijdsduur. Maar hoe onstaat resistentie? En wat is er bekend over resistentie voor anticoagulantia bij bruine ratten in Europa?

Since anticoagulant rodenticide resistance was first discovered in the United States in 1971, it has become apparent that the phenomenon is widespread. In cooperation with the Center for Disease Control, a nationwide surveillance program was initiated in 1977 to obtain statistically valid samples of rats from federally funded projects of the Urban Rat Control Program. A summary is given of the basic sampling, testing, and analysis components of this study. Problems encountered in all aspects of the first three years of the program are discussed along with results from the 40 completed samples. The 16 cities with significant Anticoagulant Resistance Problem Areas are distinguished from those in which resistance has merely been observed. Levels of resistance in various rat populations are discussed and recommendations are made in support of integrated pest management programs. Recent findings from retesting resistant rats, half of which die, are presented with regard to application of the surveillance program.

While the problem of anticoagulant resistance in commensal rodents has been well-documented from certain areas of northern Europe in recent years, this paper describes its first known occurrence in North America in 1971. Over a rural area of about 5 square miles in Johnson County, North Carolina, it was noted that attempts to control Norway rats using typical warfarin rodenticide bait were increasingly ineffective. Diphacinone was alternated with warfarin with no success. On one farm, 200 lbs of bait had been used in bait boxes in a single month. Laboratory trials on rats captured at this location demonstrated resistance. It was concluded that at this location, intensive use of anticoagulants (mostly warfarin) over a decade, in the absence of adequate sanitation and building maintenance, provided the selective agent to develop resistant populations. This likely will be repeated elsewhere in the U.S.

Between 2003 and 2010 two 3-year screening periods (I and II) were conducted to monitor the brown rat resistance to rodenticides in Flanders. Resistance was assessed by means of genetic mutations. The aim of the study was to estimate the degree of resistance in Flanders, to test whether there are differences between the 12 river basins, and whether the resistance is increasing over time. Multiple rats per location were caught.Several statistical analyses were performed on these data. On the one hand, aggregated results per location (presence/absence of resistance, defined in several ways) were analysed using logistic regression (glm), while on the other hand, mixed model logistic regression (glmm) was used on the rat-level to incorporate correlations between rats from a single location.The level of resistance differed strongly between river basins, some being almost completely resistant, while in others resistance was nearly zero. Correlation was strongest between rats within a location, but variograms also showed a strong correlation between nearby locations (The number of locations (resp. rats) per river basin ranged from 2 to 54 (resp. 2 to 151) for screening I, and from 4 to 65 (resp. 11 to 110) for screening II. Due to these small numbers, the estimate of resistance was inaccurate for some river basins. Also, evolution of resistance over time was not analysed, since data collection in both screening periods was not comparable. A more balanced follow-up monitoring program, with enough rats and locations per river basin, has been designed to answer all questions more accurately in the future.